E and P Selectins as Potential Markers in the Assessment of the Severity of Acute Pancreatitis.

OBJECTIVES: Acute pancreatitis (AP) is commonly associated with the release of adhesion molecules such as E and P selectins. We designed the present study to evaluate the role of selectins as potential markers that could reflect the severity of the disease. METHODS: One hundred fifty patients with AP constituted the patient group, whereas 70 healthy volunteers established the control group. In both groups, blood samples were taken for measurements of E selectin, P selectin, caspase-cleaved cytokeratin 18, and total soluble cytokeratin 18 levels on admission and days 1, 2, 4, and 6. RESULTS: Values of E and P selectins on admission were both elevated compared with control subjects (P < 0.01). The nonsurvivors had higher values of E selectin (P < 0.04) and P selectin (P < 0.03) on admission. Levels of E and P selectin showed positive correlation with the length of stay (P < 0.05). E selectin on admission yielded a sensitivity of 75% and 78% specificity, whereas P selectin had a sensitivity of 67% and 91% specificity. CONCLUSIONS: Selectin values in the early course of AP may play a role as indicators of overall prognosis, which may help physicians in better understanding the pathophysiology of a benign disease that may have serious and detrimental complications.

Nonalcoholic fatty liver disease: biomarkers as diagnostic tools for liver damage assessment in adult patients from Argentina.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease whose prevalence has been increasing constantly and linked to the global obesity epidemic. The NAFLD histologic spectrum ranges from simple steatosis to nonalcoholic steatohepatitis (NASH), which can progress to cirrhosis and hepatocellular carcinoma. Liver biopsy is the only reliable means to diagnose and stage NASH, but its invasive nature limits its use. Therefore, the prediction of hepatic injury by means of the development of new noninvasive tests represents a growing medical need. Our aim was to evaluate matrix deposition and cell-death markers, which correlate with liver injury in an NAFLD patient cohort. PATIENTS AND METHODS: Liver biopsies and serum from 34 NAFLD adult patients were analyzed. Histological parameters were evaluated. Matrix deposition [hyaluronic acid (HA) and tissue inhibitor of matrix metalloproteinase inhibitor-1 (TIMP-1)] and cell-death markers [cytokeratin-18 (M65) and caspase-cleaved cytokeratin-18 (M30)] were measured in serum samples. RESULTS: HA showed an association with fibrosis severity (P=0.03) and M30 with steatosis (P=0.013), inflammation (P=0.004), and fibrosis severity (P=0.04). In contrast, TIMP-1 and M65 showed no association with any histological parameter of liver injury. The evaluation of diagnostic accuracy showed good performance as less invasive markers of significant fibrosis of both HA (area under the receiver operating characteristic curve: 0.928) and M30 (area under the receiver operating characteristic curve: 0.848). CONCLUSION: Biomarkers are essential tools that may provide a quick and accurate diagnosis for patients with life-threatening NAFLD and NASH. HA and M30, together or determined sequentially, have been found to be straightforward tests that may be sufficient to predict significant fibrosis even in a primary care center of an underdeveloped country.

Assessment of Preclinical Liver and Skeletal Muscle Biomarkers Following Clofibrate Administration in Wistar Rats.

Clofibrate is a known rodent hepatotoxicant classically associated with hepatocellular hypertrophy and increased serum activities of cellular alanine aminotransferase/aspartate aminotransferase (ALT/AST) in the absence of microscopic hepatocellular degeneration. At toxic dose, clofibrate induces liver and skeletal muscle injury. The objective of this study was to assess novel liver and skeletal muscle biomarkers following clofibrate administration in Wistar rats at different dose levels for 7 days. In addition to classical biomarkers, liver injury was assessed by cytokeratin 18 (CK18) cleaved form, high-mobility group box 1, arginase 1 (ARG1), microRNA 122 (miR-122), and glutamate dehydrogenase. Skeletal muscle injury was evaluated with fatty acid binding protein 3 (Fabp3) and myosin light chain 3 (Myl3). Clofibrate-induced hepatocellular hypertrophy and skeletal muscle degeneration (type I rich muscles) were noted microscopically. CK, Fabp3, and Myl3 elevations correlated to myofiber degeneration. Fabp3 and Myl3 outperformed CK for detection of myofiber degeneration of minimal severity. miR-122 and ARG1 results were significantly correlated and indicated the absence of liver toxicity at low doses of clofibrate, despite increased ALT/AST activities. Moreover, combining classical and novel biomarkers (Fabp3, Myl3, ARG1, and miR-122) can be considered a valuable strategy for differentiating increased transaminases due to liver toxicity from skeletal muscle toxicity.

The transformation in biomarker detection and management of drug-induced liver injury.

Drug-induced liver injury (DILI) is a major concern for patients, care givers and the pharmaceutical industry. Interpretation of the serum biomarkers routinely used to detect and monitor DILI, which have not changed in almost 50 years, can be improved with recently proposed models employing quantitative systems pharmacology. In addition, several newer serum biomarkers are showing great promise. Studies in rodents indicate that the ratio of the caspase cleaved fragment of cytokeratin 18 to total K18 in serum (termed the "apoptotic index") estimates the relative proportions of apoptosis vs necrosis during drug-induced liver injury. Glutamate dehydrogenase can reliably differentiate liver from muscle injury and, when serum is properly prepared, may also detect mitochondrial toxicity as a mechanism of liver injury. MicroRNA-122 is liver-specific, but recent data suggests it can be actively released from hepatocytes in the absence of overt toxicity limiting enthusiasm for it as a DILI biomarker. Finally, damage associated molecular patterns, particularly high mobility group box 1 and its various modified forms, are promising biomarkers of innate immune activation, which may be useful in distinguishing benign elevations in aminotransferases from those that portend clinically important liver injury. These new biomarkers are already being measured in early clinical trials, but broad acceptance will require widespread archiving of serum from diverse clinical trials and probably pre-competitive analysis efforts. We believe that utilization of a panel of traditional and newer biomarkers in conjunction with quantitative systems pharmacology modelling approaches will transform DILI detection and risk management.

Drug-induced liver injury: recent advances in diagnosis and risk assessment.

Idiosyncratic drug-induced liver injury (IDILI) is a rare but potentially severe adverse drug reaction that should be considered in patients who develop laboratory criteria for liver injury secondary to the administration of a potentially hepatotoxic drug. Although currently used liver parameters are sensitive in detecting DILI, they are neither specific nor able to predict the patient's subsequent clinical course. Genetic risk assessment is useful mainly due to its high negative predictive value, with several human leucocyte antigen alleles being associated with DILI. New emerging biomarkers which could be useful in assessing DILI include total keratin18 (K18) and caspase-cleaved keratin18 (ccK18), macrophage colony-stimulating factor receptor 1, high mobility group box 1 and microRNA-122. From the numerous in vitro test systems that are available, monocyte-derived hepatocytes generated from patients with DILI show promise in identifying the DILI-causing agent from among a panel of coprescribed drugs. Several computer-based algorithms are available that rely on cumulative scores of known risk factors such as the administered dose or potential liabilities such as mitochondrial toxicity, inhibition of the bile salt export pump or the formation of reactive metabolites. A novel DILI cluster score is being developed which predicts DILI from multiple complimentary cluster and classification models using absorption-distribution-metabolism-elimination-related as well as physicochemical properties, diverse substructural descriptors and known structural liabilities. The provision of more advanced scientific and regulatory guidance for liver safety assessment will depend on validating the new diagnostic markers in the ongoing DILI registries, biobanks and public-private partnerships.

Assessment of Apoptotic Activity Dysregulation and Oxidative Stress in the Development of Epithelial Ovarian Cancer: A Case-Controlled Descriptive Analysis.

AIM: In the present study, we aimed to assess whether oxidative stress and apoptotic activity play a role in the development of epithelial ovarian cancer (EOC). METHODS: The study group included patients with EOC (n = 26) and benign ovarian tumour (BOT) (n = 25), while 30 healthy women were employed as a control group. Venous blood samples were drawn to evaluate oxidative stress parameters and serum M30/M65 antigen levels before surgery. In addition, blood samples were taken for the second time on postoperative day 8 to analyse whether the postoperative tumour load was decreased. RESULTS: When the groups were assessed regarding oxidative stress, the highest values were detected in patients with EOC. Serum M30/M65 levels were found to be higher in patients with EOC when compared to the other groups (p < 0.001). A significant decrease was determined in the M30/M65 levels of serum samples taken on postoperative day 8 from the patients in the EOC and BOT groups (p < 0.001). CONCLUSION: Our results suggest that dysregulation of apoptotic activity could be effective in the development of ovarian tumoural tissue, whereas oxidative stress could be effective in malignant transformation.

Apoptotic and anti-apoptotic seromarkers for assessment of disease severity of non-alcoholic steatohepatitis.

BACKGROUND AND STUDY AIMS: This study aimed to find out non-invasive markers for the assessment of severity of non-alcoholic steatohepatitis (NASH) in an attempt to decrease the need for liver biopsy. It also aimed to evaluate the key role of apoptosis in the pathogenesis of the disease and the suggested role of anti-apoptotic factors in therapeutic modalities and disease prognosis. PATIENTS AND METHODS: The serum levels of soluble Fas (s. Fas), s. Fas ligand, cytokeratin 18 (CK-18) fragment and Bcl-2 were measured in 80 patients and 15 non-hepatic subjects as control. The patients were divided based on histological examination of liver biopsy into three groups. Group I included 40 patients with NASH, group II had 40 patients with non-alcoholic fatty liver disease (NAFLD) non-NASH and group III had 15 non-hepatic subjects as control. Apoptosis of hepatocytes was assessed by morphological examination using a light microscope and expressed as number per square millimetre. RESULTS: There was a significant increase in the serum levels of s. Fas, s. Fas ligand and CK-18 fragments in the NASH group. The anti-apoptotic protein Bcl-2 showed significantly low levels in NASH patients. Apoptosis of hepatocytes was significantly higher in the NASH group. The degree of apoptosis was inversely correlated with the level of Bcl-2. A significant correlation between both s. Fas and CK-18 fragment with liver histology with regard to lobular inflammation and ballooning was found. CONCLUSIONS: Increased serum levels of s. Fas and CK-18 fragment in the NASH group and its correlation with the severity of disease suggested the key role of apoptosis in NASH pathogenesis which can be used for the assessment of the severity of NASH. A high level of anti-apoptotic Bcl-2 in NAFLD suggests its protective role in disease progress.

Systematic review with meta-analysis: non-invasive assessment of non-alcoholic fatty liver disease--the role of transient elastography and plasma cytokeratin-18 fragments.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) affects 15-40% of the general population. Some patients have non-alcoholic steatohepatitis (NASH) and progressive fibrosis, and would be candidates for monitoring and treatment. AIM: To review current literature on the use of non-invasive tests to assess the severity of NAFLD. METHODS: Systematic literature searching identified studies evaluating non-invasive tests of NASH and fibrosis using liver biopsy as the reference standard. Meta-analysis was performed for areas with adequate number of publications. RESULTS: Serum tests and physical measurements like transient elastography (TE) have high negative predictive value (NPV) in excluding advanced fibrosis in NAFLD patients. The NAFLD fibrosis score comprises of six routine clinical parameters and has been endorsed by current American guidelines as a screening test to exclude low-risk individuals. The pooled sensitivities and specificities for TE to diagnose F ≥ 2, F ≥ 3 and F4 disease were 79% and 75%, 85% and 85%, and 92% and 92% respectively. Liver stiffness measurement often fails in obese patients, but the success rate can be improved with the use of the XL probe. A number of biomarkers have been developed for the diagnosis of NASH, but few were independently validated. Serum/plasma cytokeratin-18 fragments have been most extensively evaluated and have a pooled sensitivity of 66% and specificity of 82% in diagnosing NASH. CONCLUSIONS: Current non-invasive tests are accurate in excluding advanced fibrosis in NAFLD patients, and may be used for initial assessment. Further development and evaluation of NASH biomarkers are needed.

Assessment of diurnal changes and confounding factors that affect circulating cell death biomarker levels: a short communication.

There is increasing use of circulating cell death biomarkers in patients and clinical trials. Knowledge of the potential noise and confounders in assays are vital for biomarker interpretation. The daily and diurnal variability and effect of menstruation and exercise on nucleosomal DNA (nDNA), total cytokeratin 18 (tK18) and apoptotic specific cytokeratin 18 (cK18) were assessed in 3 cohorts of healthy volunteers; 12 pre-menopausal women to establish the effect of menstruation, 12 men to perform exercise and 12 post-menopausal women. All 36 subjects were evaluated to establish daily and diurnal variability. Estimates of variability were derived in a linear mixed effects model and presented as the back transformed coefficient of variation (%CV). Minimal variation was seen in cK18 (11%CV) and tK18 (11%CV) but higher variability was seen in nDNA (85%CV). K18 results appeared stable throughout the day but a possible peak in nDNA was seen at 15:00. Menstruation had minimal effects but exercise led to immediate short-lived elevations in cell death biomarkers. There is no evidence of significant daily variability in K18 assays. We recommend subjects should not exercise for 6h before blood sampling.

Validation of terminal peptide of procollagen III for the detection and assessment of nonalcoholic steatohepatitis in patients with nonalcoholic fatty liver disease.

UNLABELLED: Liver biopsy is the reference standard for the detection of nonalcoholic steatohepatitis (NASH) within nonalcoholic fatty liver disease (NAFLD). The aim of this study was to identify a biomarker of NASH in patients without significant fibrosis. In all, 172 patients from two centers with biopsy-proven NAFLD were included in this study. Eighty-four patients from a single center were included as a derivation cohort and 88 patients from a second center were included as a validation cohort. Serum samples were tested for candidate markers of fibrosis and inflammation alongside hematological and biochemical markers. Among patients without advanced fibrosis, terminal peptide of procollagen III (PIIINP) was the only marker found to be associated with a histological diagnosis of NASH in both cohorts. PIIINP also correlated with the total NAFLD activity score (NAS) and its constituent components (P < 0.001). Area under receiver operating characteristic curve (AUROC) for PIIINP in discriminating between NASH and simple steatosis (SS) was 0.77-0.82 in patients with F0-2 fibrosis and 0.82-0.84 in patients with F0-3 fibrosis. PIIINP was elevated in patients with advanced fibrosis, the overwhelming majority of whom had NASH. When incorporating patients with all degrees of fibrosis from both cohorts, PIIINP was able to discriminate between patients with SS and those with NASH or advanced fibrosis with AUROC 0.85-0.87. CONCLUSION: PIIINP discriminates between SS and NASH or advanced fibrosis. The use of a single biomarker in this context will be of clinical utility in detecting the minority of patients with NAFLD who have NASH or advanced fibrosis related to NASH.

Non-invasive methods for the assessment of hepatic fibrosis: transient elastography, hyaluronic acid, 13C-aminopyrine breath test and cytokeratin 18 fragment.

BACKGROUND. In the management of chronic hepatitis C (CHC) patients, liver biopsy is the gold standard for liver fibrosis assessment despite some technical limits and risks. Non-invasive approaches have been proposed as alternative methods to evaluate structural liver damage. AIM. To investigate the diagnostic accuracy of transient elastography, 13C-aminopyrine breath test (13C-ABT), serum hyaluronic acid (HA) and cytokeratin 18 Asp396 fragment (CK-18) as non-invasive methods of liver fibrosis assessment ad their correlation to METAVIR score. MATERIAL AND METHODS. In a cohort of 57 CHC patients, liver stiffness, cumulative percentage of administered dose of 13C-aminopyrine at 120 min, serum HA and serum CK-18 concentration were determined. Diagnostic accuracy in detecting significant fibrosis (F ≥ 2), severe fibrosis (F ≥ 3) and cirrhosis (F = 4) was assessed by the area under the receiver operating characteristic curve. RESULTS. Liver fibrosis score showed a strong correlation with liver stiffness (r = 0.667; p < 0.0001) and a significant inverse correlation with 13C-ABT results (r = -0.418; p = 0.0012). A weaker correlation was found with CK18 (r = 0.329; p = 0.0126) and no correlation with HA. Areas under the curve of elastography, 13C-ABT, HA and CK18 were: 0.98, 0.75, 0.69, 0.64, respectively, for F ≥ 2; 0.97, 0.69, 0.80, 0.66, respectively, for F ≥ 3; 0.95, 0.64, 0.70, 0.56, respectively, for F = 4. CONCLUSION. Elastography has the best diagnostic accuracy for the assessment of the degree of liver fibrosis in CHC patients. Its application can provide an alternative useful tool for monitoring the disease evolution.

Assessment of non-alcoholic fatty liver disease using serum total cell death and apoptosis markers.

BACKGROUND: The diagnosis of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH) and fibrosis relies on liver biopsy. Non-invasive assessments are urgently needed. AIM: To evaluate cell apoptotic marker cytokeratin-18 M30 and total cell death markers cytokeratin-18 M65/M65ED for the assessment and monitoring of NAFLD. METHODS: A cohort of 147 patients with biopsy-proven NAFLD and 73 controls were enrolled, including 51 patients who received paired liver biopsies 36 months apart. Biomarkers were determined by enzyme-linked immunosorbent assay. RESULTS: M30, M65 and M65ED increased in a stepwise fashion in control subjects, patients with non-NASH, NAFLD and NASH (all P < 0.001). All biomarkers had similarly high accuracy over 0.9 in predicting NAFLD and moderate accuracy around 0.7 in predicting NASH. Among patients with paired liver biopsies, changes in M30, M65 and M65ED positively correlated with disease progression (rho = 0.42, 0.32 and 0.39; P = 0.002, 0.023 and 0.005 respectively), and only changes in M65 and M65ED correlated with fibrosis progression (rho = 0.29, 0.34; P = 0.038, 0.015 respectively). Both M30 and M65 had area under receiver-operating characteristics curve above 0.8 in predicting disease progression. At cut-off of 236 U/L, changes of M65ED had 88% NPV and 59% PPV to exclude and predict fibrosis progression. CONCLUSIONS: Cytokeratin-18 M30 and M65/M65ED have moderate accuracy in detecting non-alcoholic steatohepatitis. Changes in the biomarkers also correlate with histological progression. However, development of new biomarkers is still required to improve the diagnostic accuracy.

Assessment of circulating biomarkers for potential pharmacodynamic utility in patients with lymphoma.

PURPOSE: Treatment efficacy and toxicity are difficult to predict in lymphoma patients. In this study, the utility of circulating biomarkers in predicting and/or monitoring treatment efficacy/toxicity were investigated. PATIENTS AND METHODS: Circulating biomarkers of cell death (nucleosomal DNA (nDNA) and cytokeratin 18 (CK18)), and circulating FLT3 ligand, a potential biomarker of myelosuppression, were assessed before and serially after standard chemotherapy in 49 patients with Hodgkin and non-Hodgkin lymphoma. Cytokeratin 18 is not expressed in lymphoma cells so is a potential biomarker of epithelial toxicity in this setting. Tumour response was assessed before and after completion of chemotherapy by 2D and 3D computed tomography radiological response. RESULTS: Baseline nDNA level was significantly higher in all lymphoma subtypes compared with 61 healthy controls and was prognostic for progression-free survival in diffuse large B-cell lymphoma (DLBCL). Decreases in nDNA levels were observed in the first week after chemotherapy; in FL, early falls in nDNA predicted for long remission following therapy. In DLBCL, elevations in nDNA occurred in cases with progressive disease. Circulating CK18 increased within 48 h of chemotherapy and was significantly higher in patients experiencing epithelial toxicity graded >3 by Common Terminology for Classification of Adverse Events criteria. FLT3 ligand was elevated within 3-8 days of chemotherapy initiation and predicted those patients who subsequently developed neutropenic sepsis. CONCLUSION: These data suggest circulating biomarkers contribute useful information regarding tumour response and toxicity in patients receiving standard chemotherapy and have potential utility in the development of individualised treatment approaches in lymphoma. These biomarkers are now being tested within multicentre phase III trials to progress their qualification.